Antigenic polypetide sequence of factor VIII, fragments and/or epitopes there of

ABSTRACT

An antigenic polypeptide of factor VIII comprises a polypeptide included between the Glutamic Acid 1649 and Asparagine 2019, preferably between Arginine 1652 and Arginine 1917 of the polypeptide of factor VIII, or a polypeptide included between Alanine 108 and Methionine 355, or a polypeptide included between Aspartic Acid 403 and Aspartic Acid 725, or a polypeptide included between Lysine 2085 and Lysine 2249.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/BE95/00063, filed Jul. 14, 1995, whichclaims priority to Belgian application BE 9400666, filed Jul. 14, 1994.

SUBJECT OF THE INVENTION

The present invention relates to the antigenic polypeptide sequence offactor VIII, to fragments and epitopes of this sequence and to the majorparts of these epitopes, to the inhibitors which are directed againstthis sequence, its fragments, its epitopes and/or the major parts ofthese epitopes, and to the anti-inhibitors which are directed againstthe said inhibitors.

The present invention also relates to a pharmaceutical composition andto a diagnostic device comprising the abovementioned molecules.

TECHNICAL BACKGROUND UNDERLYING THE INVENTION

Recently, factor VIII preparations which have been purified from largeplasma pools by means of ion exchange chromatography, or very recentlyby means of immunoaffinity, have been made available to haemophilics inadequate quantities.

Various preparations of FVIII which have been obtained by geneticmanipulation are currently under development or under clinical trial.These FVIIIs are either intact molecules or deleted molecules (Bihoreau(1992)).

FVIII is a glycoprotein cofactor of plasma coagulation and acts at thelevel of factor X (FX) activation. Characterization of FVIII and itsmechanism of action is made more difficult because of its lowconcentration in the plasma, the size heterogeneity and its extremesensitivity to enzymic degradation. This reaction comprises theproteolysis of FX to form activated factor X (FXa=Stuart factor) andbrings into play a complex (Tenase complex) which comprises the enzyme(activated FIX or FIXa), a cofactor (activated FVIII or FVIIIa), calciumions and phospholipids.

FVIII is a protein which is so complex that, even though the sequence ofits gene has been known since 1984 (Vehar et al. 1984 Nature 312, pp.337-342), neither the complete structure of plasma FVIII (only about 50%of the protein has been sequenced) nor the precise structure of thecarbohydrates has yet been established. The DNA sequence has beenallowed to define the primary sequence of FVIII (SEQ ID NO: 21) (a rareexception to the instructions laid down by the FDA for the therapeuticproducts derived from biotechnology).

Nevertheless, subtle differences between plasma FVIII and recombinantFVIII have been identified: i.e. glycosylation, plasma half-lifefollowing infusion, etc.

FVIII is in the main synthesized in the hepatocytes. It has been clonedin mammalian cells, insect cells and yeast cells (Webb et al., 1993).These glycoproteins which are produced by biotechnological processes canexhibit differences in the structure and composition of the sugars ascompared with the natural protein. The cDNA of FVIII has also beenexpressed in transgenic sheep (Halter et al., 1993).

The cDNA encodes a polypeptide of 2351 amino acids, including the signalpeptide of 19 amino acids which is cleaved off in the endoplasmicreticulum. Post-translational modifications take place in the Golgiapparatus: i.e. glycosylation of the serines and threonines and additionof sulphate ions to the tyrosine residues. Following maturation, theprotein is subsequently secreted into the plasma in the form of 2chains, of 210 kDa (up to residue 1648) and 80 kDa (from residue 1649 toresidue 2332), which are joined by a divalent ion, with the lighterchain being linked non-covalently to the von Willebrand factor (vWf) byits N-terminal end (1 molecule of vWf per molecule of FVIII). In theplasma, this complex is stabilized by hydrophobic and hydrophilic bondsin the presence of a 50-fold excess of vWf. This latter is reported toinhibit the attachment of FVIII to phospholipids. The fact that FVIIIbinds to the platelets has been established, although the presence ofspecific receptors expressed on the surface of the platelets has notbeen clearly demonstrated (Nesheim et al., 1993). Following itsattachment to the membrane phospholipids, it is reported to unmaskhigh-affinity binding sides for FIXa (Bardelle et al., 1993).

FVIII is made up of three structural domains, A, B and C (Kaufman R J,1992; Bihoreau et al., 1992) which are arranged in the orderA1:A2:B:A3:C1:C2 (FIG. 1). The A domains possess more than 40% homologyand are also homologous to ceruloplasmin. 30% homology also existsbetween the A domains of factor V and FVIII. The C domain occurs twiceand is reported to be able to bind glycocon-jugates and phospholipidshaving a net negative charge (Kemball-Cook and Barrowcliffe (1992); Fay,P J, 1993)). It exhibits homology with lectins which are able to bind tonegatively charged phospholipids. The platelet attachment site has beenlocated in this region (C2 domain) (Foster et al., (1990)). While domainB, which represents more than 40% of the mass of FVIII, does not haveany known specific activity, it could play a subtle role in theregulation of FVIII by protecting it, for example, from the action ofthrombin. It does not possess any known homology with other proteins.

It possesses 19 glycosylation sites out of the 25 which have beenidentified in FVIII. Comparison of the amino acid sequences of human andporcine FVIII reveals major differences within this B region.Nevertheless, porcine FVIII is used effectively for treatinghaemophilics exhibiting inhibitors. These observations have led to theconstruction of an FVIII gene from which the part encoding this B regionhas been deleted and which can be used to produce a deleted recombinantFVIII which is intended for the treatment of haemophilia.

Using immunopurification, different forms of active FVIII have beenisolated which all possess a light chain of 80 kDa and whose heavy chaincan have a molecular weight of between 210 and 90 kDa. These forms arereported to be derived by progressive degradation of the C-terminal endof the heavy chain. The binding of the two chains is non-covalent andresults from a divalent metallic ion (Me++) bond between the responsibleresidues in domains A1 and A3. After formation of the activated complex(50-45 kDa) (heavy chain having accessible A2 domain) and 70 kDa (lightchain), an inactivation phase is observed, probably as a consequence ofprolonged contact with thrombin and dissociation of the 50 kDa and 45kDa fragments. FVIIIa is also inactivated by activated protein C (APC)following proteolysis of the heavy chain. This inactivation isaccelerated if the FVIIIa is attached to a phospholipid surface. Thisdown-regulation of the activity of FVIIIa is reported to depend on aphosphorylation by a platelet enzyme (Kalafatis et al., (1990)).

Most of the epitopes which are recognized by the various murinemonoclonals which have been isolated to date do not appear to be locatedin the “functional sites” of FVIII. Some epitopes have been identifiedwhich are recognized by antibodies which have an effect on the activityof FVIII (inhibition of the chromogenic and/or clotting tests).

These antigenic determinants consist of fragments 351-365 (A1domain—heavy chain), 713-740 (A2 domain), 1670-1684 (A3 domain—lightchain) (NH₂ end of the light chain) or else 2303-2332 (C2 domain—lightchain) (Foster C, (1990)), fragments 701-750 (Ware et al. (1989)),1673-1689 (Leyte et al. (1989)), 330-472, 1694-1782 (EP-0 202 853),322-740 and 2170-2322 (Scandella et al. (1992)).

The antibodies which recognize these various sites interfere,respectively, with the activation of FVIII, the binding of vWf or thebinding of phospholipids.

Other antibodies, which do not inhibit standard activity tests in vitro,can exert an influence on the behaviour of FVIII with the otherconstituents of the coagulation cascade while attaching themselves tosites in the molecule which are at a substantial distance from theactive sites. These antibodies, thus modified, can interfere with thenatural state of folding of FVIII by altering some of its properties(“allosteric model”).

These mapping experiments make use of peptides which are synthesized byFVIII gene fragments which are cloned into E. coli and only provide anapproximate idea of the location of the antigenic determinants which arerecognized by these monoclonal antibodies. Thus, the sizes of theidentified fragments range between 30 and 100 amino acids.

At present, it is necessary to crystallize a protein and analyse it withX rays in order to identify its antigenic sites unambiguously.Unfortunately, no data are available for FVIII, whose high molecularweight is a major handicap with regard to crystallization.

The antigenic regions coincide with the hydrophilic character of theseregions: the more the oligopeptide sequence is exposed to the externalmedium (situated on the surface), the more this part is capable of beingrecognized in an immune reaction. By contrast, the hydrophobic parts,which are generally situated in the interior of the protein, are notconsidered to be very antigenic.

Currently, a predominant notion among haemophilic patients, cliniciansand “fractionators” is that of having available a purified FVIII whichis devoid of all pathogenic plasma contaminants and secondary effects.

However, whether after immunopurification using murine monoclonalantibodies or after obtaining it by genetic recombination in mammaliancells, highly purified FVIII is extremely unstable for reasons which arenot apparent. In order to stabilize it, substantial quantities of humanplasma albumin are added during the course of purification, such thatthe final specific activity is of the order of 2-3 U/mg of protein. Thesame applies to rFVIII which is coexpressed with the von Willebrandfactor, which is a natural stabilizer, in CHO cells. These data appearto suggest that the purification steps exert an influence on the FVIIImolecule, with these steps being able to interfere with its naturalstate of folding, to introduce confirmational changes which are more orless stable and to reveal new potential epitopes following infusion intothe patient.

According to the authors (Ljung et al. (1992); Sultan et al., (1992);Lorenzo et al. (1992)), one of the serious complications which is seenin from 5 to 50% of the haemophilics who are given multiple therapeuticinfusions of FVIII is the appearance of antibodies (inhibitors) whichinactivate FVIII and render ineffective any subsequent injection ofFVIII.

The spontaneous appearance of autoantibodies having a pathologicalanti-FVIII activity is rare in non-haemophilics (prevalence: 10⁻⁵) andhas been reported in elderly individuals, in those exhibitingimmunological disorders and in post-partum individuals (Kessler (1991),Hultin (1991)). A multi-centre study which was carried out on 3,435haemophilic patients shows that all the age groups are affected,including patients of less than 5 years old. The majority (82%) displaya very high response (>10 BU) (Sultan et al. (1992)). While theseanti-FVIII antibodies have been reported to consist essentially of IgGantibodies of the IgG4 type, IgG2 (Gilles et al. (1993)b), IgA and IgMantibodies have also been described (Lottenburg et al. (1987)). Theyreact weakly with purified heterologous FVIII molecules from othermammals (Bennett, B et al. (1972)). At the present time, it is not knownwhat induces the appearance of the inhibitors in some haemophilics. Ifthere is an association between the severity of the deletion of the geneand the development of an immune response which no longer recognizesFVIII as a self protein, this association is only demonstrated in aminority of patients. It has not been possible to demonstrate anyspecific host susceptibility which is linked to genetic markers, suchas, for example, a preferential association with certain determinants ofthe MEC class II complex (Hoyer (1991)), without a doubt because not allthe FVIII epitopes which are recognized by specific antibodies have yetbeen determined. It also appears that the different methods of preparingFVIII could exert an influence on its structure, its physicochemicalproperties or its natural microenvironment (Vermeylen, J and Peerlinck(1991); Gomperts, et al. (1992); Peerlinck et al. (1993)). Barrowcliffeet al. (1983) have demonstrated that phospholipids protect theprocoagulatory activity from inactivation by specific human antibodies.The presence of natural anti-FVIII antibodies in 17% of healthy donors(screening carried out on 500 plasma donations) without any pathologicalsymptoms demonstrates the importance of becoming better acquainted withthe three-dimensional structural appearance assumed by physiologicalFVIII (Ciavarella and Schiavoni (1992)).

Transfusion which has been studied on mixed lymphocyte cultures, inanimal models and during clinical trials has demonstrated modificationof the immuno-modulation in the transfused subject, inducing anallo-immunization and also a down-regulation of some immune functions.It expresses itself in the form of suppressor cells, anti-idiotypeantibodies or a decrease in NK cells. It is as if a certain degree oftolerance was being induced. These effects can be reversed by infusinginterleukin 2 (IL-2) (Triulzi et al., 1990). In vitro, an inhibitoryeffect on the secretion of IL-2 as well as the proliferation ofperipheral blood mononuclear cells are obtained in the presence of acryoprecipitate or relatively impure preparations of FVIII (from 0.5 to10 U/mg of protein) (Madhok et al., 1991; Wadhwa, M et al., 1992). Theseeffects are not observed in the presence of rFVIII or FVIII which havebeen purified by immunoaffinity. This latter preparation is reported tohave an activating effect on T cells (Madhok et al., 1991). However, itis not possible to extrapolate these findings directly to an in vivosituation.

No experimental model exists which makes it possible to forecast theimmunogenicity or the immuno-modulatory effect of the FVIIIpreparations, or the susceptibility of the host, before they have beenadministered clinically. This model becomes an absolute necessity in theface of the increase in the frequency of the appearance of anti-FVIIIantibodies in current clinical trials which make use of FVIIIpreparation which are of very high specific activity and which have beenobtained either by immunopurification or by DNA manipulation techniques(Seremetis et al. (1991)). In addition, Aledorf (1993) has demonstratedthat when these two types of preparation are used in naive subjects whohave not previously been transfused (PUPS), an inhibitor prevalence isobserved which amounts to up to 27%.

STATE OF THE ART

Patients who develop an anti-FVIII immune response find themselves in aserious situation which necessitates the use of severe, aggressive andexcessively expensive measures. One of the most frequently employedtechniques is to swamp the organism with regular injections of very highdoses of FVIII (from 100 to 200 U/kg/day) (Ewing et al. (1988)) inassociation with a concentrated prothrombin complex (FEIBA) (Bonn'sprotocol), a procedure which effectively reduces the level of inhibitorsin the blood (Sultan et al. 1986). In addition, this type of treatmenthas to be continued for a very long time (Lian et al., 1989). Trialscarried out using smaller doses of FVIII have met with a certain degreeof success in patients whose anti-FVIII antibody levels are much lower(Gruppo, (1991)).

An alternative approach is to use FVIII from a non-human species such asthe pig, which FVIII is not neutralized by the anti-FVIII of the patientand enables haemostasis to take place. While a multi-centre study hasshown the advantages of such a treatment, it has also demonstrated thatanti-porcine FVIII antibodies are formed (Lozier (1993); Moreau et al.(1993); Hay and Bolton-Maggs (1991); Clyne et al. (1992)). Activatedfactor VIII, obtained by recombinant DNA technology, has also beenemployed as an alternative means for achieving coagulation in patientswho exhibit inhibitors (Ingerslev et al. (1991)).

Recently, a profitable strategy (Nilsson et al. (1990)) for reducing thelevel of inhibitors has consisted in subjecting patients to anextracorporeal circulation to enable solid-phase absorption of the totalIgG to be effected on protein A while at the same time treating thepatients with cytostatic agents such as cyclophosphamide.

The infusion of polyvalent intravenous immunoglobulins (IVIG), whereappropriate combined with an immunosuppressive treatment, has been foundto be relatively effective, although the reason for this effectivenessis still not fully established. Various hypotheses involving feed-backinhibition of IgG synthesis, stimulation of IgG clearance or activationof T suppressor cells have been advanced (Bloom (1992)). An interestingexplanation is that these commercial intravenous immunoglobulins mightcontain antibodies which are able to react with the variable parts(idiotypes) of the anti-FVIII antibodies and neutralize theseantibodies. It is suggested that this anti-idiotype activity might bespecific to each donor and could be synergistic within an IgG pool(Dietrich et al. (1992)).

Unfortunately, none of these approaches has been found to besatisfactory in terms of safety, efficiency and cost.

OBJECTS OF THE INVENTION

The present invention is aimed at obtaining an antigenic polypeptidesequence of factor VIII, and fragments and epitopes of this sequence,whose purpose is to improve the diagnosis and/or therapy of immunedisorders, in particular those induced by inhibitors of FVIII andinhibitors of the binding of FVIII to the von Willebrand factor (vWf)and/or to membrane phospholipids (PL).

Another object of the invention is aimed at obtaining inhibitors whichexhibit an immunoaffinity with this antigenic polypeptide sequence, itsfragments and/or its epitopes, whose purpose is also to improve thediagnosis and/or therapy of immune disorders.

A supplementary object is aimed at obtaining anti-inhibitors, inparticular antibodies, which are directed against the abovementionedsaid inhibitors and whose purpose is to improve the diagnosis and/ortherapy of immune disorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 diagrammatically depicts the polypeptide sequence of factor VIII.

FIG. 2 depicts the hydrophilicity graph of the A3 sequence of factorVIII renumbered from 1 to 371 amino acids (surface value for each aminoacid).

FIG. 3 depicts the flexibility graph for this A3 sequence of factorVIII.

FIG. 4 depicts the accessibility graph for this A3 sequence of factorVIII.

FIG. 5 depicts the general graph representing the sum of the valuesdefined in Graphs 2 to 4.

FIG. 6 depicts the demonstration of anti-factor VIII antibodies in mousesera using an ELISA test.

CHARACTERISTIC ELEMENTS OF THE INVENTION

The present invention relates to the antigenic polypeptide sequence offactor VIII and/or fragments of this sequence, as described by Verhar etal. (Nature, Vol. 312, p. 339 (1984)).

The “polypeptide sequence of factor VIII” is understood to be thenatural human or animal sequence, which may be glycosylated and whichhas been obtained by purification from pools of plasma, in particularCohn fraction I, by synthesis and/or by genetic manipulation (that isincluding a sequence from which portions which are not involved in themechanism of blood coagulation may have been deleted) of factor VIII.

The present invention relates, in particular, to the antigenicpolypeptide sequence of factor VIII which lacks fragments alanine322-serine 750, leucine 1655-arginine 1689, lysine 1694-proline 1782 andaspartic acid 2170-tyrosine 2332.

The present invention relates, in particular, to the antigenicpolypeptide sequences A1, A2, A3 and C (C1 and C2) of factor VIII.

In the remainder of the text, the amino acids will be represented bytheir three-letter abbreviations or by the single-letter symbol, asidentified in the table below.

Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn NMethionine Met M Aspartic Asp D Phenylalanine Phe F acid Cysteine Cys CProline Pro P Glutamine Gln Q Serine Ser S Glutamic Glu E Threonine ThrT acid Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr YIsoleucine Ile I Valine Val V

A first embodiment of the invention relates to the antigenic polypeptidesequence A3 of factor VIII, and to fragments and/or epitopes of thissequence. The said sequence is contained between glutamic acid 1649 andasparagine 2019, preferably between arginine 1652 and arginine 1917 orbetween arginine 1803 and arginine 1917, of the polypeptide sequence offactor VIII as published by Verhar et al. (Nature, vol. 312, p 339(1984)) and Toole et al. (Nature, vol. 312, pp. 342-347 (1984)).

Preferably, the fragments of the said sequence are contained betweenarginine 1652 and arginine 1696, preferably between arginine 1652 andarginine 1689, between threonine 1739 and aspartic acid 1831 or betweenglutamic acid 1885 and arginine 1917.

The invention also relates to the sequence epitopes of these fragments,in particular:

the epitope contained between arginine 1652 and tyrosine 1664, definedby the following sequence:

Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr SEQ ID No:1: 1                  5                       10

the epitope contained between aspartic acid 1681 and arginine 1696,defined by the following sequence:

Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg SEQ IDNo:2:  1               5                   10                  15

the epitope contained between threonine 1739 and tyrosine 1748, definedby the following sequence:

SEQ ID No:3: Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr 1               5                   10

the epitope contained between asparagine 1777 and phenylalanine 1785,defined by the following sequence:

Asn Gln Ala Ser Arg Pro Tyr Ser Phe SEQ ID No:4:  1               5

the epitope contained between glutamic acid 1794 and tyrosine 1815,defined by the following sequence:

Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro SEQ IDNo:5:  1               5                   10                  15 AsnGlu Thr Lys Thr Tyr              20

the epitope contained between methionine 1823 and aspartic acid 1831,defined by the following sequence:

Met Ala Pro Thr Lys Asp Glu Phe Asp SEQ ID No:6:  1               5

the epitope contained between glutamic acid 1885 and phenylalanine 1891,defined by the following sequence:

Glu Thr Lys Ser Trp Tyr Phe SEQ ID No:7:  1               5

the epitope contained between glutamic acid 1893 and alanine 1901,defined by the following sequence:

Glu Asn Met Glu Arg Asn Cys Arg Ala SEQ ID No:8:  1               5

the epitope contained between aspartic acid 1909 and arginine 1917,defined by the following sequence:

Asp Pro Thr Phe Lys Glu Asn Tyr Arg SEQ ID No:9:  1               5

Advantageously, the said sequence, its specific fragments and itsepitopes exhibit an antigenic characteristic which is illustrated byappended FIGS. 2 to 5.

Another preferred embodiment of the invention relates to antigenicpolypeptide sequence A1 of factor VIII and fragments and/or epitopes ofthis sequence.

Preferably, the fragments of the said sequence are contained betweenalanine 108 and methionine 355, preferably between alanine 108 andglutamine 228.

The invention also relates to the sequence epitopes of these fragments,in particular:

the epitope contained between alanine 108 and valine 128, defined by thefollowing sequence:

Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys SEQ IDNo:10:  1               5                   10                  15 GluAsp Asp Lys Val              20

the epitope contained between glutamic acid 181 and leucine 192, definedby the following sequence:

SEQ ID No:11: Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 1               5                   10

the epitope contained between aspartic acid 203 and glutamine 218,defined by the following sequence:

Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln SEQ IDNo:12:  1               5                   10                  15

the epitope contained between aspartic acid 327 and methionine 355,defined by the following sequence:

Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu SEQ IDNo:13:  1               5                   10                  15 AlaGlu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met             20                      25

Another preferred embodiment of the invention relates to the antigenicpolypeptide sequence A2 of factor VIII and fragments and/or epitopes ofthis sequence.

Preferably, the fragments of the said sequence are contained betweenaspartic acid 403 and aspartic acid 725, preferably between histidine693 and aspartic acid 725.

The invention also relates to the sequence epitopes of these fragments,in particular:

the epitope contained between aspartic acid 403 and lysine 425, definedby the following sequence:

Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg SEQ IDNo:14:  1               5                   10                  15 IleGly Arg Lys Tyr Lys Lys                          20

the epitope contained between valine 517 and arginine 527, defined bythe following sequence:

SEQ ID No:15: Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg 1               5                   10

the epitope contained between histidine 693 and glycine 701, defined bythe following sequence:

His Asn Ser Asp Phe Arg Asn Arg Gly SEQ ID No:16:  1               5

the epitope contained between serine 710 and aspartic acid 725, definedby the following sequence:

Ser Cys Asp Lys Asn Thr Gly Asp Tyr Try Gly Asp Ser Tyr Glu Asp SEQ IDNo:17:  1               5                   10                  15

A final preferred embodiment of the invention relates to the antigenicpolypeptide sequence C of factor VIII, and fragments and/or epitopes ofthis sequence. Preferably, the fragments of the said sequence arecontained between lysine 2085 and lysine 2249, preferably between lysine2085 and glycine 2121.

The invention also relates to the sequence epitopes of these fragments,in particular:

the epitope contained between lysine 2085 and phenylalanine 2093,defined by the following sequence:

Lys Thr Gln Gly Ala Arg Gln Lys Phe SEQ ID No:18:  1               5

the epitope contained between aspartic acid 2108 and glycine 2121,defined by the following sequence:

Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly SEQ ID No:19: 1               5                   10

the epitope contained between glycine 2242 and lysine 2249, defined bythe following sequence:

Gly Val Thr Thr Gln Gly Val Lys SEQ ID No:20:  1               5

The invention also relates to the major parts of the said epitopes orthe said fragments, that is to say to the portions of the sequences ofthe said epitopes which contain the amino acids tyrosine and histidine,which unexpectedly display a particularly high affinity towardsinhibitors of factor VIII. Preferably, these major parts contain thesaid amino acid tyrosine or histidine linked to at least two otheridentical or different amino acids.

These sequences, these fragments and these epitopes, in particular themajor parts of the epitopes or the fragments, are particularlyadvantageously characterized by high hydrophilicity, such as describedby Parker, Guo and Hodges (Biochemistry 25, pp 5425-5432 (1986)),considerable flexibility, such as described by Karplus and Schultz(Naturwissenschaften 72, p 212 (1985)) and considerable accessibility,such as described by Janin (Nature 277, pp 491-492 (1979)) (see FIGS. 2to 5).

These fragments and these epitopes are, in particular, exposed on thesurface of the factor VIII protein and exhibit a pronounced antigeniccharacteristic.

Advantageously, the said polypeptide sequence, its fragments, itsepitopes and/or these major parts of the said fragments or the saidepitopes are also independently immunogenic (that is to say they areimmunogenic even without being complexed with a protein of large sizesuch as BSA, haemocyanin, etc.), and preferably exhibit animmunoaffinity within inhibitors of factor VIII, such as anti-factorVIII antibodies, and/or exhibit an immunoaffinity for the receptors ofthe T lymphocytes and/or B lymphocytes.

This sequence, these fragments, these epitopes and/or the major parts ofthe said fragments or the said epitopes induce an immune reaction(antibody synthesis) when they are injected into a rabbit.

These characteristics are particularly pronounced in the case of theepitopes SEQ ID No: 2 and SEQ ID No: 5, which comprise sequences whichare relatively “long” in amino acids, i.e. comprise 16 and 22 aminoacids, respectively.

These sequences are therefore characterized by substantialimmunogenicity towards monoclonal and polyclonal antibodies.

However, these sequences are sufficiently short to be readily obtainedby synthesis.

As an example, peptides Asp 1681-Arg 1696 and Asp 327-Met 355 weresynthesized in order to demonstrate the presence of anti-factor VIIIantibodies in mouse sera using an ELISA test.

The free peptides (not coupled to a carrier protein) were injected intotwo BALB/C mice in accordance with the following protocol:

-   -   day 0 100 μg of peptide emulsified in incomplete Freund's        adjuvant are injected intramuscularly.    -   days 7, 14, 21 and 28: immunization with 50 μg of peptide.

A sample of blood is withdrawn on each day before the injection.Polystyrene microtitration plates (NUNC) are saturated with apreparation of plasma factor which is diluted with the aid of 40 IU/ml.50 μl volumes of increasing dilutions (1/60, 1/300 and 1/600) of mouseantisera are added to the wells. Following incubation and washing, thepresence of anti-factor VIII antibodies is demonstrated by adding 50 μlof a 1/5000 dilution of a rabbit anti-mouse IgG antibody which islabelled with biotin. Following incubation and washing, the wells areincubated with 50 μl of avidin/peroxidase (1/2500) and washed, with 100μl of OPD finally being added to the wells. The optical density ismeasured at 490 nm. The results of the ELISA are presented in appendedFIG. 6 (EX1, EX2 and a sample, termed BLC, which serves as the blank).

The present invention also relates to the conformational epitopes whichcomprise at least two different fragments of the said sequence, at leasttwo sequence epitopes and/or at least two major parts of the saidepitopes or the said different fragments according to the invention andidentified above.

The conformational epitopes are made up of two or more differentportions of a polypeptide sequence, which portions are located inproximity to each other when the protein is folded in its tertiary orquaternary structure.

These epitopes are capable of being “recognized” (that is to say ofexhibiting an immunoaffinity), preferably simultaneously, withinhibitors of factor VIII, in particular B lymphocytes (by way of themajor histocompatibility locus (MHC I and/or II)) and/or anti-factorVIII antibodies (Scandella et al., Blood 76, p 437 (1990)).

Preferably, the said sequence, the said fragments, the said epitopesand/or the major parts of the said epitopes or the said fragments arecomplexed with a carrier protein or a carrier peptide, such as BSA orhaemocyanin, in such a way as to form a complex exhibiting a morepowerful immunogenicity.

Another aspect of the present invention relates to an inhibitor offactor VIII which exhibits an immunoaffinity with the antigenicpolypeptide sequence according to the present invention, with fragmentsand epitopes of this sequence, with major parts of the said epitopes orthe said fragments and/or with the complex according to the invention.

An inhibitor is understood to mean any biological molecule or cell whichintervenes with and/or against factor VIII and is capable of giving riseto immune disorders.

In particular, such an inhibitor can be an anti-factor VIII monoclonalor polyclonal antibody (gamma-globulin) or antibody fragment (such asthe hypervariable Fab portion of the said antibody) which inactivatesthe said factor VIII and/or which inhibits the binding of factor VIII tothe von Willebrand factor and/or to membrane phospholipids.

Advantageously, the said inhibitors are synthesized by a “chimaeric”animal which comprises a human immune system, such as an hu-SCID mouseproducing human antibodies.

SCID (severe combined immunodeficient) mice are mice which exhibit adeficiency in functional B and T lymphocytes due to dysfunction of therecombination of the genes which are responsible for the antigenicreceptors. The immune system of the SCID mice can be reconstituted usingimmunocompetent cells of human origin which are derived from foetalorgans or peripheral blood (Mosler et al. (1988)).

Once reconstituted, these hu-SCID mice produce human antibodies eitherspontaneously or after immunization.

There does not appear to be any dramatic cross reactivity between humanfactor VIII and murine factor VIII (Kessler, 1991).

The peripheral blood lymphocytes are taken from several types of donor:non-haemophilic volunteers, haemophilics who lack inhibitors which canbe detected by standard methods, haemophilics who exhibit substantialinhibitor levels and donors who are producing autoantibodies.

This model is employed in two types of study. Firstly, the mice arereconstituted with cells from a single donor, and it is possible tocompare the antigenicity of several factor VIII preparations once thereproducibility of the system has been verified.

On the other hand, it is possible, using this model, to obtain and studyan anti-factor VIII response at the clonal level.

Study of the specific monoclonal response of the B cells is veryimportant since this enables the sequential and conformational epitopesof factor VIII to be identified precisely. The B cells are cultured,cloned in the presence or absence of anti-CD40 antibodies, from thespleens of mice which are producing anti-factor VIII antibodies, or elsetransformed in the presence of EBV virus. The anti-CD40 antibodiesrecognize a membrane antigen and activate the B cells in the presence ofa fibroblast line (Banchereau et al. 1991)). It is consequently possibleto envisage using these immunodominant epitopes as a possible target forimmunotherapy.

Determination of the MHC class I and class II markers which are carriedby the B lymphocyte clones makes it possible to analyze the immuneresponse of the anti-factor VIII antibodies at the genetic level andthereby to follow recognition by the specific T cells. This is also anexcellent method for seeing whether there is a risk factor associatedwith this pathology.

The BALB/C mice which are selected for preparing the anti-factor VIIIMabs are firstly injected on three occasions, at 2 week intervals, witha solution of recombinant factor VIII (rFVIII). This type of preparationhas the advantage of containing a high-purity factor VIII at elevatedconcentration together with a minimum of contaminating proteins. Fourdays after the last injection, the splenocytes are fused with the cellsfrom a mouse myeloma (SP207) (van Snick and Coulie (1982)). Thehybridomas which are producing anti-factor VIII antibodies are selectedby the ELISA technique, using polystyrene plates on which rFVIII haspreviously been insolubilized. The hybridoma supernatants containing theanti-factor VIII antibodies are cloned by the limiting dilutiontechnique and then cultured in vitro.

The antibodies are purified from these supernatants by means ofchromatography.

The ELISA technique is used to quantify, and to determine the lightchain (k or 1) and the subclass (IgG1, IgG2a, IgG2b or IgG3) of theanti-factor VIII Mabs.

The epitopes which are recognized on the factor VIII molecule aredetermined by means of the immunotransfer technique using solutions ofnative factor VIII or factor VIII which has been cleaved enzymicallywith thrombin.

The ability of each of the anti-factor VIII Mabs which have beenproduced to inhibit function is evaluated both by a coagulation method(Bethesda method) (Kasper et al. (1975)) and by a chromogenic methodwhich is based on the ability which is possessed by factor X, which hasbeen activated by association with factor VIII and activated factor IX,to transform a colourless substrate into a coloured substrate (Svendsenet al. (1984)).

The cell lines which produce human monoclonal anti-factor VIIIantibodies are derived from human B lymphocytes which are taken from theabdominal cavity of SCID mice which have been immunized with differentbatches of factor VIII after reconstitution of the immune system of theanimals with human lymphocytes. The B lymphocytes are cultured in thepresence of fibroblast cells which express a receptor for theimmunoglobulin Fc moiety, to which is attached a monoclonal anti-CD40antibody. These cells, which have been activated by polymerization ofthe CD40 receptor, are then infected and immortalized with Epstein-Barrvirus (Kozbor, (1981)). The cell lines which produce the sought-afterantibodies can then be subcloned.

Another aspect of the invention relates to an anti-inhibitor which ischaracterized in that it is directed against the said previouslydescribed factor VIII inhibitor.

An anti-inhibitor which is directed against the factor VIII inhibitor isunderstood to mean any biological molecule and/or cell which is capableof interfering with the said inhibitor in such a way as to ensure itsinactivation.

Preferably, such an anti-inhibitor is an anti-anti-factor VIII idiotype(monoclonal or polyclonal) antibody or antibody fragment.

Advantageously, these anti-inhibitors which are directed against thefactor VIII inhibitors are synthesized by a “chimaeric” animalexhibiting a human immune system, such as an hu-SCID mouse.

Only mice which produce less than 10 μg/ml residual immunoglobulins areused for the experiments.

The model is developed using peripheral leucocytes which are derivedfrom volunteers who have been immunized against tetanus.

The reconstitution is effected by means of a single i.p. injection offrom 15 to 20.10⁶ mononuclear cells of human origin. These cells areobtained after centrifuging peripheral blood (approximately 200 ml) on aFicoll/Hypaque gradient. From twelve to twenty mice can be reconstitutedfrom one single donor. The production of human immunoglobulins ismeasured as a function of time.

The anti-anti-factor VIII idiotype antibodies are purified from a poolof starting plasma which is assembled from voluntary donations from atleast 7200 donors in order to increase the probability of findinganti-idiotype antibodies by means of immunoaffinity using humananti-factor VIII antibodies which are covalently attached to a Sepharosecolumn or attached by means of an Fc moiety to a protein G column.Following fractionation, by means of the Cohn-Oncley method, twoIgG-rich fractions, Fr II and Fr III, are obtained. They will serve asthe starting preparation for purifying the anti-idiotype antibodies.These monoclonal antibodies will be obtained from B cells which havebeen taken from haemophilic patients. These cells have initiallyproliferated in SCID mice and have been transformed into secretory cellcultures by the EBV virus. Use of these human monoclonal antibodiesmakes it possible to avoid introducing non-human proteins into thetherapeutic preparations. These preparations are evaluated, by means ofdetailed immunochemical analysis, for their efficiency in neutralizingthe inhibitors which are derived from the largest possible number ofhaemophilic patients. Several physical (treatment with UVCF radiation),thermal and/or chemical (for example using a solvent/detergent) viralinactivation steps are introduced into the purification process in orderto ensure the greatest possible degree of viral safety.

The idiotype which is peculiar to the human antibodies is analyzed bysequencing the variable moiety of the molecule. These data are of theutmost importance because they are of great value both in diagnosing andregulating the production of anti-factor VIII antibodies.

Up to the present time, the source of the antibodies which are requiredfor preparing antigen/antibody complexes has been autologous, that is tosay the patient himself was supplying the antibodies. It has recentlybecome clear that normal individuals, having normal levels ofcirculating factor VIII, produce anti-factor VIII antibodies whoseactivity in the plasma is limited by corresponding anti-idiotypeantibodies. Anti-factor VIII antibodies which have been prepared from agammaglobulin pool can advantageously replace the autologous source.

It is also possible to obtain human B cells which have been transformedwith the EBV virus and which produce inhibitors from haemophilic ornon-haemophilic patients. Four lines have thus been obtained, with oneof these lines recognizing the light chain of factor VIII. SCID micehave been repopulated with inhibitor-secreting B cells derived fromhaemophilic or non-haemophilic patients. Production is stimulated byinjecting plasma factor VIII and recombinant factor VIII. It istherefore possible to obtain continuous in-vitro cultures which areproducing the said inhibitors. Anti-anti-factor VIII idiotype antibodiescan also be produced continuously using this technique.

Another aspect of the invention relates to a pharmaceutical compositionwhich comprises an element which is selected from the group consistingof the said antigenic polypeptide sequence of factor VIII, fragments andepitopes of this sequence and/or major parts of the said epitopes or thesaid fragments, an inhibitor of factor VIII which is directed againstthem, an anti-inhibitor which is directed against the said inhibitor,and/or a mixture of these.

Another aspect of the invention relates to a diagnostic and/orpurification device such as a diagnostic kit or a chromatography column(such as described by Ezzedine et al. (1993)), which comprises anelement which is selected from the group consisting of the antigenicpolypeptide sequence according to the invention, fragments and epitopesof this sequence and/or the major parts of the said epitopes or the saidfragments, the complex according to the invention, an inhibitor which isdirected against them, an anti-inhibitor which is directed against thesaid inhibitor, and/or a mixture of these.

The purification device can therefore consist of a chromatography columnsuch as described by Ezzedine et al. (1993) which comprises the sequenceof factor VIII, fragments and epitopes of this sequence and/or the majorparts of the said fragments or epitopes, which are attached to the solidphase of the chromatography column.

A physiological liquid (such as serum), which is derived from a patientand which comprises inhibitors of factor VIII, is then caused to passthrough this chromatography column, with the said inhibitors (forexample antibodies) becoming attached specifically to the said factorVIII, the said fragments, the said epitopes or the said major parts.

Following elution, it is possible to collect the said inhibitors bycausing them to react with anti-inhibitors (anti-anti-factor VIIIidiotype antibodies).

It is also possible to characterize the anti-anti-factor VIII idiotypeantibodies which are present in a serum by causing these anti-inhibitorsto be passed through a chromatography column on which inhibitors offactor VIII have been attached to the solid phase.

A final aspect of the invention relates to the use of the pharmaceuticalcomposition according to the invention for preparing a medicament to beused for preventing and/or treating immune disorders, in particularthose which are induced by inhibitors of factor VIII, inhibitors of thebinding of factor VIII and the von Willebrand factor (vWF) and/orinhibitors of the binding of factor VIII to membrane phospholipids.

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1. An isolated antigenic fragment of the human Factor VIII polypeptideof SEQ ID NO: 21, said fragment comprising at least 7 amino acids of ahuman Factor VIII fragment selected from the group consisting of a humanFactor VIII fragment extending from arginine 1652 to arginine 1696inclusive, a human Factor VIII fragment extending from threomine 1739 totyrosine 1748 inclusive (SEQ ID NO: 3), a human Factor VIII fragmentextending from asparagine 1777 to phenylalanine 1785 inclusive (SEQ IDNO: 4), a and a human Factor VIII fragment extending from glutamic acid1885 to arginine 1917 inclusive.
 2. The isolated antigenic fragmentaccording to claim 1, wherein said human Factor VIII fragment comprisesan epitope selected from the up consisting of: a human Factor VIIIfragment extending from arginine 1652 to tyrosine 1664 (SEQ ID No: 1), ahuman Factor VIII fragment extending from threonine 1739 to tyrosine1748 (SEQ ID No: 3), a human Factor VIII fragment extending fromasparagine 1777 to phenylalanine 1785 (SEQ ID No: 4), a human FactorVIII fragment extending from glutamic acid 1885 to phenylalanine 1891(SEQ ID No: 7), a human Factor VIII fragment extending from glutamicacid 1893 to alanine 1901 (SEQ ID No: 8), and a human Factor VIIIfragment extending from aspartic acid 1909 to arginine 1917 (SEQ ID No:9).
 3. The isolated antigenic fragment according to claim 1, whereinsaid antigenic polypeptide comprises tyrosine or histidine.
 4. Anisolated conformational epitope comprising at least two different humanFactor VIII fragments of claim 2, wherein said fragments are positionedin proximity to each other when the protein is folded in its tertiary orquaternary structure to form a conformational epitope which isrecognized by an inhibitor of Factor VIII selected from the groupconsisting of B lymphocytes, MHC I proteins, MHC II proteins, andanti-Factor VIII antibodies.
 5. An isolated conformational epitopecomprising at least two different epitopes from a fragment of the humanFactor VIII polypeptide of SEQ ID NO: 21 wherein said fragment isselected from the group consisting of a human Factor VIII fragmentextending from arginine 1652 to arginine 1696 inclusive, a human FactorVIII fragment extending from threonine 1739 to aspartic acid 1831,inclusive, and a human Factor VIII fragment extending from glutamic acid1885 to arginine 1917 inclusive.
 6. A complex, comprising a carrierprotein or a carrier peptide linked to the fragment of claim 1 or theconformational epitope of claim 5, wherein said complex has higherimmunogenicity than said polypeptide of claim
 1. 7. A pharmaceuticalcomposition comprising at least the antigenic fragment of claim 1, orthe conformational epitope of claim 5 and an acceptable pharmaceuticalvehicle.
 8. The complex of claim 6, wherein said carrier protein or saidcarrier peptide are bovine serum albumin or hemocyanin.
 9. An isolatedpolypeptide, consisting of a fragment of the human Factor VIIIpolypeptide of SEQ ID NO: 21, wherein said fragment is selected from thegroup consisting of a human Factor VIII fragment between arginine 1652and arginine 1696 inclusive, a human Factor VIII fragment betweenglutamic acid 1885 and arginine 1917 inclusive, and a fragmentcomprising at least 7 amino acids thereof, wherein said polypeptide isantigenic.
 10. A pharmaceutical composition, comprising the antigenicpolypeptide of claim 9 and an acceptable pharmaceutical vehicle.
 11. Acomplex, comprising a carrier protein or a carrier peptide linked to theantigenic polypeptide of claim 9, wherein said complex has higherimmunogenicity than said polypeptide of claim
 9. 12. The complex ofclaim 11, wherein said carrier protein or said carrier peptide arebovine serum albumin or hemocyanin.
 13. A conformational epitopecomprising at least two different human Factor VIII fragments of claim9, wherein said fragments are positioned in proximity to each other whenthe protein is folded in its tertiary or quaternary structure to form aconformational epitope which is recognized by an inhibitor of FactorVIII selected from the group consisting of B lymphocytes, MHC Iproteins, MHC II proteins, and anti-Factor VIII antibodies.
 14. Anisolated polypeptide, consisting of a fragment of the human Factor VIIIpolypeptide of SEQ ID NO: 21, wherein said human Factor VIII fragmentconsists of an epitope selected from the group consisting of: a humanFactor VIII fragment contained between arginine 1652 and tyrosine 1664(SEQ ID No: 1), a human Factor VIII fragment contained between threonine1739 and tyrosine 1748 (SEQ ID No: 3), a human Factor VIII fragmentcontained between asparagine 1777 and phenylalanine 1785 (SEQ ID No: 4),a human Factor VIII fragment contained between glutamic acid 1794 andtyrosine 1815 (SEQ ID No: 5), a human Factor VIII fragment containedbetween methionine 1823 and aspartic acid 1831 (SEQ ID No: 6), a humanFactor VIII fragment contained between glutamic acid 1885 andphenylalanine 1891 (SEQ ID No: 7), a human Factor VIII fragmentcontained between glutamic acid 1893 and alanine 1901 (SEQ ID No: 8),and a human Factor VIII fragment contained between aspartic acid 1909and arginine 1917 (SEQ ID No: 9), wherein said polypeptide is antigenic.15. A pharmaceutical composition, comprising the antigenic polypeptideof claim 14 and an acceptable pharmaceutical vehicle.
 16. A complex,comprising a carrier protein or a carrier peptide linked to theantigenic polypeptide of claim 14, wherein said complex has higherimmunogenicity than said polypeptide of claim
 9. 17. The complex ofclaim 16, wherein said carrier protein or said carrier peptide arebovine serum albumin or hemocyanin.
 18. A conformational epitopecomprising at least two different human Factor VIII fragments of claim14, wherein said fragments are positioned in proximity to each otherwhen the protein is folded in its tertiary or quaternary structure toform a conformational epitope which is recognized by an inhibitor ofFactor VIII selected from the group consisting of B lymphocytes, MHC Iproteins, MHC II proteins, and anti-Factor VIII antibodies.
 19. Anisolated polypeptide, consisting of a fragment of the human Factor VIIIpolypeptide of SEQ ID NO: 21, wherein said fragment is threonine 1739 toaspartic acid 1831, wherein said polypeptide is antigenic.